The osteoblast or bone forming cell s=is a cell of mesodermal origin that once fully differentiated produces the bone extracellular matrix and eventually mineralizes it. Alterations in osteoblast differentiation and/or function occur in several crippling genetic diseases and some degenerative diseases such as osteoporosis. The objective of this application is to use a combination of genetic, biochemical and molecular approaches centered around the osteocalcin gene, an osteoblast specific gene, to better understand the mechanisms governing osteoblast differentiation and function. Osteocalcin is a mineral binding protein of unknown function secreted y the osteoblast into the bone extracellular matrix at the time of bone mineralization. To understand osteocalcin function and thereby the function of the osteoblast during bone mineralization, we have generated an osteocalcin-deficient mouse through homologous recombination in embryonic stem cells. We plan to use this mouse model to study the functions of osteocalcin. We also intend to use these mutant mice to study osteocalcin interaction with other proteins secreted by osteoblasts in the bone extracellular matrix. To begin to decipher the mechanisms controlling osteoblast differentiation we will look for genes located upstream of the osteocalcin genes that control their osteoblast specific pattern of expression. For that purpose we will use a combination of transgenic mice analysis, DNA transfection experiments, DNA binding assays, and molecular cloning to identify, characterize, and eventually clone a cDNA coding for an osteoblast specific transcription factor. This dual study of the function and regulation of expression of a major biosynthetic product of the osteoblast should allow us to answer basic questions of cell biology centered around the function of the osteoblast in physiological and pathological circumstances.